Direct whole-mount imaging of fungal spores by energy-filtering transmission electron microscopy

Whole-mount fungal spores were examined by energy-filtering transmission electron microscopy. Conidia of Penicillium species and Ustilaginoidea virens were suspended in distilled water and directly placed on a glow-discharged formvar-coated copper grid. Energy-filtered images were taken from 0 to 100eV loss regions. Due to their considerable inherent thickness, their globose morphology was evident. In zero-loss images, the fungal spores appeared to have higher contrast in general, showing darker periphery than unfiltered images. Most spores in zero-loss images exhibited almost homogeneous electron density across the spores. The contrast was partially inversed in low-loss images where more details of the outer cell wall ornamentations of spores could be discerned than zero-loss images. As obvious advantages of whole-mount spore imaging, it allows for ensuring two-dimensional images with higher spatial resolution than light microscopy and conventional scanning electron microscopy. If a higher resolution is needed to observe fungal surface structures such as fimbriae and rodlet layers, or discriminate an outer sheath enveloping spores, whole-mount spore imaging can be employed to unravel structural details.     

Image contrast in the backscattered electron mode in scanning electron microscopy and microtomography

The problem of image contrast production in the backscattered electron (BSE) mode in a scanning electron microscope (SEM) in bulk and film structures is discussed. The considerable influence of the parameters of a semiconductor detector on the image contrast is shown. Calculations for contrast in dependence on the composition of target sections, the energy of primary electrons, and the signal detection technique are presented.     

Beam deceleration for block-face scanning electron microscopy of embedded biological tissue

The beam deceleration (BD) method for scanning electron microscopes (SEM) also referred to as "retarding" was applied to back-scattered electron (BSE) imaging of the flat block face of a resin embedded biological specimen under low accelerating voltage and low beam current conditions. BSE imaging was performed with 0-4 kV of BD on en bloc stained rat hepatocyte. BD drastically enhanced the compositional contrast of the specimen and also improved the resolution at low landing energy levels (1.5-3 keV) and a low beam current (10 pA). These effects also functioned in long working distance observation, however, stage tilting caused uncorrectable astigmatism in BD observation. Stage tilting is mechanically required for a FIB/SEM, so we designed a novel specimen holder to minimize the unfavorable tilting effect. The FIB/SEM 3D reconstruction using the new holder showed a reasonable contrast and resolution high enough to analyze individual cell organelles and also the mitochondrial cristae structures (~5 nm) of the hepatocyte. These results indicate the advantages of BD for block face imaging of biological materials such as cells and tissues under low-voltage and low beam current conditions.     

Electron channelling contrast imaging of dislocations in a conventional SEM

Abstract

Dislocations in shock loaded tantalum single crystals were imaged using both transmission electron microscope (TEM) and electron channelling contrast image (ECCI) in a scanning electron microscope with a conventional backscattered electron detector. The results were compared with backscattered electron intensity profiles across dislocations calculated via the dynamic theory of electron diffraction. A one-to-one correspondence between ECCI and TEM is established. High voltage and low index reflections should be used to obtain the highest dislocation contrast and greatest imaging depth.     

Monte Carlo方法模拟低能电子在Al中的散射

基于文献[1]的工作,电子在固体中的弹性散射用Mott微分截面计算;非弹性散射分为单电子激发和等离子激发并由Streitwolf、Gryzinski及Quinn的截面描述.模拟了低能电子在Al块样及薄膜中的散射过程,对不同能量低能电子作用下Al的背散射系统、能谱又透射系数作了计算,结果与实验符合较好.也对背散射电子、低能损背散射电子表面分布作了计算,结果表明低能损背散射电子具有较好的空间分辨率.     

多晶体二次电子的角度分布

 根据二次电子发射的主要物理过程,推导了内二次电子到达多晶表面并逸出的几率的角度分布、斜射入多晶的高能原电子产生的二次电子的角度分布和由背散射电子产生的二次电子的角度分布。同时,推导了高能原电子轰击多晶产生的二次电子的角度分布公式,该公式表明多晶的二次电子遵循余弦分布,且与原电子的入射角无关。分析结果表明: 在内二次电子最大逸出深度范围内,如果由射入多晶的原电子和背散射电子产生的内二次电子数是常数, 则多晶的二次电子的角度分布遵循余弦分布;如果由射入多晶的原电子和背散射电子产生的内二次电子数越来越少,则多晶的二次电子发射角度分布随出射角减少得比出射角的余弦值更慢;如果由射入多晶的原电子和背散射电子产生的内二次电子越来越多,则多晶的二次电子发射角度分布随出射角减少得比出射角的余弦值更快。     

Methodology for processing backscattered electron images. Application to Aguada archaeological paints

Scanning electron microscopy is a powerful technique in several fields of science and technology. In particular it is an important complement in the characterization of materials for which X-ray analysis is not possible. Such is the case of thin paint layers on ceramic pots, in which, even for low incident energies, the electron interaction volume can be greater than the paint thickness—in addition to the problem arising from similar compositions. With the aim of complementing other common techniques used in compositional materials characterization, in this work, an image-processing software has been developed, which implements a new methodology for the treatment of backscattered electron (BSE) images in order to bring to evidence small mean atomic number contrasts, usually imperceptible to human eye. The program was used to study black and white pigments of ceramic pieces belonging to the Ambato style of “Aguada” culture (Catamarca province, Argentina, IV–XII centuries AD). Although the BSE images acquired for these samples showed no apparent contrast between sherd and black and white pigments, through image-processing algorithms using different space filters, chemical contrast between regions has been brought to evidence with a minor detail loss. This has been accomplished by applying a smoothing filter, after which the main routine for contrast enhancement reveals details in the grey-level region of interest; finally, a filter for edge enhancement permits to recover some details lost in the previous steps, achieving satisfactory results for the painted sherd samples analyzed. In order to validate the mean atomic number differences found between each pigment and the ceramic body, X-ray diffraction diagrams have been refined with Rietveld method using the software DIFFRACplus Topas^®, arriving to mineralogical differences which agree with the results obtained. As a consequence of this study, the program developed has proven to be a suitable tool for routine analysis of samples with slight chemical contrast.     

高入射能量下的金属二次电子发射模型

基于高入射能量电子产生二次电子发射的物理过程, 分别对高入射能量电子产生的真二次电子和背散射电子的概率进行理论分析与建模. 利用Bethe能量损失模型和内二次电子逸出概率分布, 推导出高入射能量电子产生有效真二次电子发射的系数与入射能量的关系式; 根据高入射能量电子在材料内部被吸收的规律, 推导出高入射能量电子产生背散射电子的系数与入射能量之间的关系式. 结合两者得到高入射能量下金属的二次电子发射模型. 利用该模型计算得到典型金属材料Au, Ag, Cu, Al的二次电子发射系数, 理论计算结果与采用Casino软件模拟金属内部散射过程得到的数值模拟结果相符. 关键词： 二次电子发射 高入射能量 金属表面 散射过程     

低能电子显微术及其应用

低能电子显微术是新发展起来的一种显微探测技术。它的特点是利用低能（１－３０ｅＶ）电子的弹性背散射使表面实空间实时成像，具有高的横向（１５ｎｍ）和纵向（原子级）分辩率，且易与低能电子衍射及其他电子显微术相结合。近年来它已有效地应用于金属和半导体表面的形貌观测、表面相变、吸附、反应及生长过程的研究。     

Backscattered electron imaging for reduced charging of moisturized corn starch granules: implications for versatile imagery of hygroscopic powder specimens

Charging artifacts and surface features of corn starch granules were investigated by scanning electron microscopy. Three types of industrial waxy corn starch granules with different levels of moisture content (0, 10.3, and 24.2%) were prepared and subjected to both secondary electron imaging and backscattered electron imaging. There were no significant charging artifacts in secondary electron images at 3 or 5 kV. However, imaging at higher magnifications and accelerating voltages much lower than 3 kV ranging from 0.1 to 1 kV did not show well-resolved structures. At higher accelerating voltages than 5 kV, charging was manifested as excessive brightness at specific areas and alteration of bright and dark lines in the direction of the raster pattern in secondary electron images of all the types of specimens tested. As the accelerating voltage increased up to 30 kV in secondary electron images, the charging also increased. Meanwhile, no charging was detected in all the backscattered electron images taken at different accelerating voltages. As the accelerating voltage increased in backscattered electron images, the resolution increased with less depth of focus. Consistent results were found in all the types of corn starch granules assayed in this study. These results suggest that a simple and rapid morphological analysis of moisturized starches can be performed by backscattered electron imaging without considerable heat drying of starches. Concomitantly, it allows for imposing a higher accelerating voltage to ensure better image resolution, facilitating morphological characterization of diverse starch granules as they are in native states.     

金属二次电子发射系数表达式

根据二次电子发射的主要物理过程,推导出二次电子发射系数和单位背散射电子产生的内二次电子数与原电子产生的内二次电子数之比、原电子入射能量、参数、最大二次电子发射系数、背散射系数之间的关系式。根据实验结果,给出了2~10 keV比率的表达式。根据推导的关系式,用实验数据分别计算出6种金属的平均参数。发现6种金属的平均参数都近似为一个常数11.89(eV)0.5。根据推导的关系式和计算的参数,推导出以背散射系数、原电子入射能量和最大二次电子发射系数为变量的二次电子发射系数通式。用该通式计算出二次电子发射系数,并与相应的实验值进行了比较,最后成功地推导出金属2~10 keV的二次电子发射系数通式。     

Imaging of growth banding of minerals using 2-stage sectioning: application to accessory zircon

In the past, investigations of mineral growth were routinely carried out by the application of light-microscopic methods and, later on, by the production of single crystal sections and their documentation using specific imaging techniques (CL, BSEI, etc.). In the present work, a method is described which enables the precise sectioning of elongate crystals parallel and perpendicular to their longest axis. By examining backscattered electron images of parallel and perpendicular sections of the same grain, growth of all faces may be evaluated without major geometric correction. The new technique is applied to zircon crystals of a granitoid exposed in the southwestern Bohemian Batholith, Austria. For the studied zircon population, pyramidal and prism development during crystal growth is worked out very clearly by the imaging procedure. Besides its significance in crystal studies, the introduced method could also find a use in material science for the growth study of synthetic mineral phases.     

Visualising impregnated chitosan in Pinus radiata early wood cells using light and scanning electron microscopy

Chitosan, a deacetylated product of an abundant naturally occurring biopolymer chitin, has been used in a range of applications, particularly in food and health areas, as an antimicrobial agent. In the work reported here Pinus radiata wood was impregnated with chitosan as an environmentally compatible organic biocide (Eikenes et al., 2005a, Eikenes et al., 2005b) to protect wood against wood deteriorating microorganisms and to thus prolong the service life of wooden products. We developed sample preparation techniques targeted to visualise impregnated chitosan within wood tissues using light microscope and field-emission scanning electron microscope (FE-SEM). Sections were viewed with the light microscope without staining with a dye as well as after staining with the dye toluidine blue. Light microscopy was also undertaken on sections that had been stained with 1% aqueous osmium tetroxide (OsO₄). For SEM observations, the sections were treated with OsO₄ and then examined with the FE-SEM, first in the secondary electron imaging mode (SEI) and then in the backscattered electron imaging (BEI) mode, imaging the same areas of a section in both SEI and BEI modes. The preparation techniques employed and the combined use of light and scanning electron microscopy provided valuable complementary information, revealing that chitosan had penetrated into the cavities (cell lumens, intercellular spaces) of all sizes present within wood tissues and had also impregnated early wood cell walls. The information obtained is discussed in relation to its importance in further development of chitosan formulations and refinement of impregnation technologies to optimise chitosan impregnation into and distribution within wood tissues as well as in assessing chitosan efficacy.     

Morphology and antimony segregation of spangles on batch hot-dip galvanized coatings

Spangles produced by batch hot-dip galvanizing process have a rougher surface and a greater surface segregation of alloying element compared with those in continuous hot-dip galvanizing line (CGL), owing to the cooling rate of the former is much smaller than that of the later. Therefore, typical spangles on a batch hot-dipped Zn-0.05Al-0.2Sb alloy coating were investigated. The chemical, morphological characterization and identification of the phases on the spangles were examined by scanning electron microscopy (SEM), backscattered electron imaging (BSE), atomic force microscopy (AFM), energy dispersive spectroscopy (EDS) and X-ray diffraction analysis (XRD). The results showed that the coating surface usually exhibited three kinds of spangles: shiny, feathery and dull spangle, of which extensively antimony surface segregation was detected. The nature of precipitate on the coating surface was identified as β-Sb₃Zn₄, The precipitated β-Sb₃Zn₄ particles distributed randomly on the shiny spangle surface, both β-Sb₃Zn₄ particles and dentritic segregation of antimony dispersed in the dendritic secondary arm spacings of the feathery spangle and on the whole dull spangle surface. The dentritic segregation of antimony and precipitation of Sb₃Zn₄ compound are discussed by a proposed model.     

Reconstructing the energy spectra of backscattered electrons with allowance for the spectrometer’s response function

The inverse problem of reconstructing the true spectrum of electrons backscattered from massive and layered targets with allowance for the spread function of the toroidal sector energy analyzer and for the response function of the spectrometer??s electron detector is solved. We present the results from studying the energy spectra of a number of homogeneous samples and film-on-substrate systems obtained at different energies of the irradiating electron beam at normal incidence of the electrons on the surface, and at a 45° angle of backscattered electron detection.     

Case studies on the application of high-resolution electron channelling contrast imaging – investigation of defects and defect arrangements in metallic materials

In 1967, Coates discovered the electron channelling contrast of backscattered electrons (BSEs) in scanning electron microscopy, and by this the possibility to investigate arrangements of lattice defects in deformed microstructures of materials. Since that time, a straightforward development of the scanning electron microscopes as well as of the electron channelling contrast technique took place. Nowadays, the performance of scanning electron microscopes is high enough that the resolution of electron channelling contrast imaging (ECCI) micrographs is comparable with conventional bright field transmission electron microscopy (TEM) micrographs. In the first part of the present paper, a historical review on the development of the ECCI technique starting from its discovery more than 45 years ago up to the combination with other advanced methods of scanning electron microscopy like electron backscatter diffraction or high-resolution selected area channelling patterning in the last few years is given. Major important investigations using this technique for the visualization of individual lattice defects like stacking faults (SFs) and dislocations or dislocation arrangements are chronologically summarized. The second part demonstrates that nowadays, ECCI micrographs taken in high-resolution scanning electron microscopes can be called high-resolution ECCI (HR-ECCI). It is shown that the resolution of individual SFs and dislocations in the HR-ECCI micrographs is comparable to that of conventional TEM (about 15 nm defect image width). Furthermore, the paper is demonstrating that HR-ECCI micrographs can be obtained for various types of materials after different mechanical loadings and different grain sizes ranging from large grain size of 500 μm (cast steel) down to less than 2 μm (γ-TiAl).     

Monte Carlo方法计算低能电子作用下固体背散射电子发射及空间分布

应用Monte Carlo方法,对能量E₀≤5keV低能电子作用下固体Al、Cu、Ag、Au的背散射电子发射及表面空间分布作了计算.模型应用Mott散射截面及修正的Bethe方程分别描述低能电子在固体中的弹性和非弹性散射.计算了背散射电子能量分布、表面空间分布、深度分布和角分布规律及特征,还计算分析了背散射电子角分布与深度分布、表面空间分布及能量分布之间的关系,系统地描述了背散射电子的发射及分布规律.     

Characterization of semiconductor detectors of (1–30)-keV monoenergetic and backscattered electrons

Semiconductor detectors of backscattered electrons are basic elements of all modern scanning electron microscopes. Their quality is determined by the properties of planar p-n junctions and the parameters of the protective layer on the detector surface. The main characteristics of semiconductor detectors are considered, their response functions are calculated, and the threshold signal cutoff energies are found both for a monoenergetic electron beam and for detection of the total energy spectrum of backscattered electrons. The experimental results are in good agreement with the computational model data.     

三维PIC数值模拟中二次发射的实现

探讨了电子撞击金属表面产生二次电子的理论,介绍了二次电子发射系数、非弹性背散射电子能量分布等重要物理参数的计算式。根据相关的理论及公式,编写了三维PIC数值模拟程序的二次发射模块, 建立相应的数值模型进行模拟。模拟所得的二次电子发射系数及非弹性背散射电子的能量分布等主要参数与理论值一致,验证了模拟过程的正确性。     

Contribution of backscattered electrons to the total electron yield produced in collisions of 8–28 keV electrons with tungsten

It is shown experimentally that under energetic electron bombardment the backscattered electrons from solid targets contribute significantly (∼80%) to the observed total electron yield, even for targets of high backscattering coefficients. It is further found that for tungsten (Z = 74) with a backscattering coefficient of about 0.50, about 20% of the total electron yield is contributed by the total secondary electrons for impact energies in the range of 8–28 keV. The yield of true backscattered electrons at normal incidence (η ₀), total secondary electrons (δ) and the total electron yield (δ _tot) produced in collisions of 8–28 keV electrons with W have been measured and compared with predictions of available theories. The present results indicate that the constant-loss of primary electrons in the target plays a significant role in producing the secondary electrons and that it yields a better fit to the experiment compared to the power-law.